The experiments proposed in the projects of this application involve the study ofthe immune responses directed to the viral vector capsid and the transgene product in AAV-mediated gene transfer. Thus, the Vector Core is an essential constituent of this proposal, as it will provide support to the experimental activities outlined. All the projects require several different transgenes, reporter genes, and expression cassettes to be packaged in alternate AAV serotypes 1 through 9, together with other capsid variants, including variants with mutations at surface-exposed tyrosine residues, or with insertions of immunomodulatory viral peptides into the N-terminus of VP2, a vector capsid protein. Furthermore, the projects involve in vitro (cell culture) and in vivo (small animal models) experiments. Accomplishing the goals of each individual project will need a high degree of flexibility in terms of quality, number, and scale of each individual AAV preparation. It should be noted that differences in capsid structure of newly isolated serotypes and/or capsid mutants may require fine-tuning ofthe purification and formulation protocols, therefore it is preferable to have expertise in AAV vector production within the program rather than relying on contract organizations. During the previous funding period we established a Vector Core with proven production capability (~8E15 vector genomes produced), able to achieve economies of scale, and, most importantly, providing reliable vectors with a high level of purity and potency. The Vector Core will continue to produce AAV vectors with consistent and comparable quality in the new proposal using a helper virus-free transfection system and gradient centrifugation purification techniques, to support proposed studies in vitro and in animal models. The Core will produce, purify, quantify, aliquot, and store the vectors and provide the requested quality controls, depending on users'request. Quality ofthe vector will be carefully monitored to maintain titer, purity, and potency. Additionally, as in the previous funding period, the Core will manage other services for the projects, including characterization of AAV vector preparations, DNA plasmid production, peptide library resuspension and storage, and other activities that serve the scope ofthe proposed application.